Transcripts initiated in the region immediately upstream from the human dihydrofolate reductase gene but transcribed from the opposite strand have been identified in human cells. These divergent upstream transcripts are polyadenylated, start at 89 base pairs upstream from the dihydrofolate reductase major initiation site, and are composed of two species of 5.0 and 3.8 kilobases in length. We have isolated complementary DNA clones derived from the divergent transcripts and identified a 3.5-kilobase open reading frame in one of these clones. Computer-assisted sequence analyses have shown that there is no significant sequence homology between the putative amino acid sequence and known protein sequences. This arrangement of two divergent transcriptional units suggests that the expression of these two genes may be regulated by a bidirectional promoter with common sequence elements.